The immune cell infiltrate in the tumour microenvironment of phaeochromocytomas and paragangliomas.

Emerging evidence suggests the composition of the tumour microenvironment (TME) correlates with clinical outcome and that each tumour type has a unique TME including a variable population of inflammatory cells. We performed immunohistochemistry on 65 phaeochromocytoma and paraganglioma (PPGL) tumour samples with 20 normal adrenal medulla samples for comparison. The immune cells assessed were macrophages, lymphocytes and neutrophils, and we compared the proportion of infiltration of these immune cells with clinical and histopathological factors. There was a higher proportion of immune cells in tumour tissue compared to non-neoplastic adrenal medulla tissue, with a predominance of macrophages. There was a higher proportion of M2:M1 macrophages and T-helper lymphocytes in aggressive tumours compared to indolent ones. For SDHB-associated tumours, there was a higher proportion of M2 macrophage infiltration, with higher M2:M1 in aggressive SDHB PPGLs compared to indolent tumours. These data demonstrate that immune cells do infiltrate the TME of PPGLs, confirming that PPGLs are immunologically active tumours. Differences in the TME of PPGLs were observed between aggressive and indolent tumours. These differences could potentially be exploited as an aid in predicting tumour behaviour.

Mechanical loading inhibits cartilage inflammatory signalling via an HDAC6 and IFT-dependent mechanism regulating primary cilia elongation.

OBJECTIVE: Physiological mechanical loading reduces inflammatory signalling in numerous cell types including articular chondrocytes however the mechanism responsible remains unclear. This study investigates the role of chondrocyte primary cilia and associated intraflagellar transport (IFT) in the mechanical regulation of interleukin-1ß (IL-1ß) signalling. DESIGN: Isolated chondrocytes and cartilage explants were subjected to cyclic mechanical loading in the presence and absence of the cytokine IL-1ß. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were used to monitor IL-1ß signalling whilst Sulphated glycosaminoglycan (sGAG) release provided measurement of cartilage degradation. Measurements were made of HDAC6 activity and tubulin polymerisation and acetylation. Effects on primary cilia were monitored by confocal and super resolution microscopy. Involvement of IFT was analysed using ORPK cells with hypomorphic mutation of IFT88. RESULTS: Mechanical loading suppressed NO and PGE2 release and prevented cartilage degradation. Loading activated HDAC6 and disrupted tubulin acetylation and cilia elongation induced by IL-1ß. HDAC6 inhibition with tubacin blocked the anti-inflammatory effects of loading and restored tubulin acetylation and cilia elongation. Hypomorphic mutation of IFT88 reduced IL-1ß signalling and abolished the anti-inflammatory effects of loading indicating the mechanism is IFT-dependent. Loading reduced the pool of non-polymerised tubulin which was replicated by taxol which also mimicked the anti-inflammatory effects of mechanical loading and prevented cilia elongation. CONCLUSIONS: This study reveals that mechanical loading suppresses inflammatory signalling, partially dependent on IFT, by activation of HDAC6 and post transcriptional modulation of tubulin.

Chondrocyte expansion is associated with loss of primary cilia and disrupted hedgehog signalling.

Tissue engineering-based therapies targeting cartilage diseases, such as osteoarthritis, require in vitro expansion of articular chondrocytes. A major obstacle for these therapies is the dedifferentiation and loss of phenotype accompanying chondrocyte expansion. Recent studies suggest that manipulation of hedgehog signalling may be used to promote chondrocyte re-differentiation. Hedgehog signalling requires the primary cilium, a microtubule-based signalling compartment, the integrity of which is linked to the cytoskeleton. We tested the hypothesis that alterations in cilia expression occurred as consequence of chondrocyte dedifferentiation and influenced hedgehog responsiveness. In vitro chondrocyte expansion to passage 5 (P5) was associated with increased actin stress fibre formation, dedifferentiation and progressive loss of primary cilia, compared to primary (P0) cells. P5 chondrocytes exhibited ~50 % fewer cilia with a reduced mean length. Cilia loss was associated with disruption of ligand-induced hedgehog signalling, such that P5 chondrocytes did not significantly regulate the expression of hedgehog target genes (GLI1 and PTCH1). This phenomenon could be recapitulated by applying 24 h cyclic tensile strain, which reduced cilia prevalence and length in P0 cells. LiCl treatment rescued cilia loss in P5 cells, partially restoring hedgehog signalling, so that GLI1 expression was significantly increased by Indian hedgehog. This study demonstrated that monolayer expansion disrupted primary cilia structure and hedgehog signalling associated with chondrocyte dedifferentiation. This excluded the possibility to use hedgehog ligands to stimulate re-differentiation without first restoring cilia expression. Furthermore, primary cilia loss during chondrocyte expansion would likely impact other cilia pathways important for cartilage health and tissue engineering, including transforming growth factor (TGF), Wnt and mechanosignalling.

The primary cilium influences interleukin-1ß-induced NFκB signalling by regulating IKK activity.

The primary cilium is an organelle acting as a master regulator of cellular signalling. We have previously shown that disruption of primary cilia assembly, through targeting intraflagellar transport, is associated with muted nitric oxide and prostaglandin responses to the inflammatory cytokine interleukin-1ß (IL-1ß). Here, we show that loss of the primary cilium disrupts specific molecular signalling events in cytosolic NFκB signalling. The induction of cyclooxygenase 2 (COX2) and inducible nitrous oxide synthase (iNOS) protein is abolished. Cells unable to assemble cilia exhibit unaffected activation of IκB kinase (IKK), but delayed and reduced degradation of IκB, due to diminished phosphorylation of inhibitor of kappa B (IκB) by IKK. This results in both delayed and reduced NFκB p65 nuclear translocation and nuclear transcript binding. We also demonstrate that heat shock protein 27 (hsp27), an established regulator of IKK, is localized to the ciliary axoneme and cellular levels are dramatically disrupted with loss of the primary cilium. These results suggest that the primary cilia compartment exerts influence over NFκB signalling. We propose that the cilium is a locality for regulation of the molecular events defining NFκB signalling events, tuning signalling as appropriate.

Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes.

OBJECTIVE: Hedgehog signalling is mediated by the primary cilium and promotes cartilage degeneration in osteoarthritis. Primary cilia are influenced by pathological stimuli and cilia length and prevalence are increased in osteoarthritic cartilage. This study aims to investigate the relationship between mechanical loading, hedgehog signalling and cilia disassembly in articular chondrocytes. METHODS: Primary bovine articular chondrocytes were subjected to cyclic tensile strain (CTS; 0.33 Hz, 10% or 20% strain). Hedgehog pathway activation (Ptch1, Gli1) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5) expression were assessed by real-time PCR. A chondrocyte cell line generated from the Tg737(ORPK) mouse was used to investigate the role of the cilium in this response. Cilia length and prevalence were quantified by immunocytochemistry and confocal microscopy. RESULTS: Mechanical strain upregulates Indian hedgehog expression and activates hedgehog signalling. Ptch1, Gli1 and ADAMTS-5 expression were increased following 10% CTS, but not 20% CTS. Pathway activation requires a functioning primary cilium and is not observed in Tg737(ORPK) cells lacking cilia. Mechanical loading significantly reduced cilium length such that cilia became progressively shorter with increasing strain magnitude. Inhibition of histone deacetylase 6 (HDAC6), a tubulin deacetylase, prevented cilia disassembly and restored mechanosensitive hedgehog signalling and ADAMTS-5 expression at 20% CTS. CONCLUSIONS: This study demonstrates for the first time that mechanical loading activates primary cilia-mediated hedgehog signalling and ADAMTS-5 expression in adult articular chondrocytes, but that this response is lost at high strains due to HDAC6-mediated cilia disassembly. The study provides new mechanistic insight into the role of primary cilia and mechanical loading in articular cartilage.

The majority of adrenocorticotropin receptor (melanocortin 2 receptor) mutations found in familial glucocorticoid deficiency type 1 lead to defective trafficking of the receptor to the cell surface.

CONTEXT: There are at least 24 missense, nonconservative mutations found in the ACTH receptor [melanocortin 2 receptor (MC2R)] that have been associated with the autosomal recessive disease familial glucocorticoid deficiency (FGD) type 1. The characterization of these mutations has been hindered by difficulties in establishing a functional heterologous cell transfection system for MC2R. Recently, the melanocortin 2 receptor accessory protein (MRAP) was identified as essential for the trafficking of MC2R to the cell surface; therefore, a functional characterization of MC2R mutations is now possible. OBJECTIVE: Our objective was to elucidate the molecular mechanisms responsible for defective MC2R function in FGD. METHODS: Stable cell lines expressing human MRAPalpha were established and transiently transfected with wild-type or mutant MC2R. Functional characterization of mutant MC2R was performed using a cell surface expression assay, a cAMP reporter assay, confocal microscopy, and coimmunoprecipitation of MRAPalpha. RESULTS: Two thirds of all MC2R mutations had a significant reduction in cell surface trafficking, even though MRAPalpha interacted with all mutants. Analysis of those mutant receptors that reached the cell surface indicated that four of six failed to signal, after stimulation with ACTH. CONCLUSION: The majority of MC2R mutations found in FGD fail to function because they fail to traffic to the cell surface.

Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.

We studied the ability of heat shock, DnaJ-like-1 (HSJ1) proteins (which contain DnaJ and ubiquitin-interacting motifs) to reduce polyglutamine-mediated inclusion formation. The experiments demonstrated that expression of heat shock protein 70 (hsp70), hsp40, HSJ1a, and HSJ1b significantly reduced protein inclusion formation in a model of spinal and bulbar muscular atrophy (SBMA). HSJ1a also mediated a significant decrease in the number of inclusions formed in a primary neuronal model of protein aggregation. Studies to elucidate the mechanisms underlying these reductions showed that hsp70 and hsp40 increased chaperone-mediated refolding. In contrast, expression of HSJ1 proteins did not promote chaperone activity but caused an increase in ubiquitylation. Furthermore, HSJ1a was associated with a ubiquitylated luciferase complex, and in the presence of HSJ1a but not an HSJ1a UIM mutant (HSJ1a-deltaUIM) there was a reduction in luciferase protein levels. Together these results show that HSJ1 proteins mediated an increase in target protein degradation via the ubiquitin-proteasome system (UPS). We also found that the expression of HSJ1a significantly decreased the number of neurons containing inclusions in an in vivo model of polyglutamine disease. These findings indicate that targeted modification of the UPS to facilitate degradation of misfolded proteins may represent a highly effective therapeutic avenue for the treatment of polyglutamine disease.

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK.

Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.

Hsp40 Molecules That Target to the Ubiquitin-proteasome System Decrease Inclusion Formation in Models of Polyglutamine Disease.

We studied the ability of heat shock, DnaJ-like-1 (HSJ1) proteins (which contain DnaJ and ubiquitin-interacting motifs) to reduce polyglutamine-mediated inclusion formation. The experiments demonstrated that expression of heat shock protein 70 (hsp70), hsp40, HSJ1a, and HSJ1b significantly reduced protein inclusion formation in a model of spinal and bulbar muscular atrophy (SBMA). HSJ1a also mediated a significant decrease in the number of inclusions formed in a primary neuronal model of protein aggregation. Studies to elucidate the mechanisms underlying these reductions showed that hsp70 and hsp40 increased chaperone-mediated refolding. In contrast, expression of HSJ1 proteins did not promote chaperone activity but caused an increase in ubiquitylation. Furthermore, HSJ1a was associated with a ubiquitylated luciferase complex, and in the presence of HSJ1a but not an HSJ1a UIM mutant (HSJ1a-ΔUIM) there was a reduction in luciferase protein levels. Together these results show that HSJ1 proteins mediated an increase in target protein degradation via the ubiquitin-proteasome system (UPS). We also found that the expression of HSJ1a significantly decreased the number of neurons containing inclusions in an in vivo model of polyglutamine disease. These findings indicate that targeted modification of the UPS to facilitate degradation of misfolded proteins may represent a highly effective therapeutic avenue for the treatment of polyglutamine disease.

Neuronal DnaJ proteins HSJ1a and HSJ1b: a role in linking the Hsp70 chaperone machine to the ubiquitin-proteasome system?

The heat-shock protein 70 chaperone machine is functionally connected to the ubiquitin-proteasome system by the co-chaperone CHIP. In this article, we discuss evidence that the neuronal DnaJ proteins HSJ1a and HSJ1b may represent a further link between the cellular protein folding and degradation machineries. We have demonstrated that HSJ1 proteins contain putative ubiquitin interaction motifs and can modulate the cellular processing of rhodopsin, a protein that is targeted for degradation by the proteasome when it is misfolded.

An atypical phenotype of macular and peripapillary retinal atrophy caused by a mutation in the RP2 gene.

AIMS: To determine the molecular basis and describe the phenotype of an atypical retinal dystrophy in a family presenting with bilateral, progressive central visual loss. METHODS: Family members were examined. Investigations included Goldman perimetry, electrophysiology, and autofluorescence imaging. Candidate gene screening was performed using SSCP and sequence analysis. The proband's lymphoblastoid cells were examined for protein expression. RESULTS: Fundal examination of the proband, his mother, and brother revealed peripapillary and macular atrophy. Autosomal dominant retinal dystrophy was suspected, but less severe disease in the mother led to screening for mutations in X linked genes. A 4 bp microdeletion in exon 3 of the RP2 gene, segregating with disease, was identified. No RP2 protein expression was detected. CONCLUSION: The distinct phenotype in this family, caused by this frameshifting mutation in RP2, broadens the phenotypic spectrum of X linked retinitis pigmentosa. The absence of RP2 protein suggests that loss of protein function and not novel gain of function could account for the atypical phenotype. A definitive diagnosis of X linked retinitis pigmentosa permits appropriate genetic counselling with important implications for other family members. Clinicians should have a low threshold for screening RP2 in families with retinal dystrophy, including posterior retinal disease, not immediately suggestive of X linked inheritance.

Unfolding retinal dystrophies: a role for molecular chaperones?

Inherited retinal dystrophy is a major cause of blindness worldwide. Recent molecular studies have suggested that protein folding and molecular chaperones might play a major role in the pathogenesis of these degenerations. Incorrect protein folding could be a common consequence of causative mutations in retinal degeneration disease genes, particularly mutations in the visual pigment rhodopsin. Furthermore, several retinal degeneration disease genes have recently been identified as putative facilitators of correct protein folding, molecular chaperones, on the basis of sequence homology. We also consider whether manipulation of chaperone levels or chaperone function might offer potential novel therapies for retinal degeneration.

Identification and characterization of a human mitochondrial homologue of the bacterial co-chaperone GrpE.

We have identified a novel human cDNA with a predicted protein sequence that has 28% amino acid identity with the E. coli Hsp70 co-chaperone GrpE and designated it HMGE. Even with this low level of amino acid identity the human sequence could be efficiently modelled on the X-ray structure of the E. coli protein, suggesting that there may be significant functional conservation. Indeed, HMGE expressed in E. coli as a GST fusion protein co-purified with the E. coli Hsp70 protein DnaK in the absence of ATP. DnaK could be released from the GST-HMGE with a Mg-ATP wash. Subcellular fractionation and immunocytochemistry studies using antisera raized against HMGE show that it is a mitochondrial protein. In contrast to studies of rat GrpE, however, HMGE also appears to bind the constitutive cytosolic Hsp70, Hsc70, in addition to mitochondrial Hsp70, Mt-Hsp70. We have previously shown that Hsc70 nucleotide-exchange is rate limiting in the presence of the DnaJ-protein, HSJ1b. However, HMGE was found to inhibit the HSJ1b-enhanced Hsc70 ATPase activity and may mediate this inhibition by binding the DnaJ-protein, HSJ1b. This is the first description of a direct interaction between a DnaJ protein and GrpE-like protein. These studies suggest that the structure of GrpE has been conserved throughout evolution and that the conserved structure can interact with several forms of Hsp70, but that HMGE cannot form part of the reaction cycle for cytosolic Hsc70.

Mutations in the N-terminus of the X-linked retinitis pigmentosa protein RP2 interfere with the normal targeting of the protein to the plasma membrane.

The X-linked retinitis pigmentosa (XLRP) gene, RP2, codes for a novel 350 amino acid protein of unknown function. We have identified putative sites for N-terminal acyl modification by myristoylation and palmitoylation in the RP2 protein. The RP2 protein is expressed ubiquitously in human tissues at relatively low levels (0.01% of total protein) and has a predominantly plasma membrane localization in cultured cells, as would be expected if the protein was subject to dual N-terminal acylation. Furthermore, mutagenesis of residues potentially required for N-terminal acylation prevents targeting of RP2 to the plasma membrane and the N-terminal 15 amino acids of the protein appear to be sufficient for this targeting. Our data suggest that the protein is dually acylated and that the palmitoyl moiety is responsible for targeting of the myristoylated protein from intracellular membranes to the plasma membrane. The effect of two mutations, which have been reported as causes of XLRP, R118H and DeltaS6, were investigated. The R118H mutation does not affect the normal plasma membrane localization of RP2; in contrast, the DeltaS6 mutation interferes with the targeting of the protein to the plasma membrane. Therefore, the DeltaS6 mutation may cause XLRP because it prevents normal amounts of RP2 reaching the correct cellular locale, whereas the R118H mutation is in a region of the protein that is vital for another aspect of RP2 function in the retina.